Retroviridae are unique among virus families in that one virion packages two full-length copies of its genome. However, each infectious event generates only one provirus;thus, retroviruses are pseudodiploid. We do not fully understand the benefit of pseudodiploidy, and we propose to examine the benefit of packaging two RNAs in one virion. Additionally, the exact mechanisms that retroviruses use to package two RNAs are not currently understood. We have performed experiments examining the mechanisms of copackaged RNA partner selection and have demonstrated that the DIS sequence plays an important role in this process. In our experimental system, we can increase or decrease heterozygous virus formation by manipulating the DIS sequences of the two viruses. These results suggest that Gag recognizes and packages an RNA dimer rather than two monomers. We have developed an imaging method to directly examine RNA in the viral particles (single-virion analysis). By this method, we have determined that most HIV-1 particles contain one RNA dimer. Using various approaches, we have also concluded that HIV-1 Gag mostly packages dimeric RNA, which initiates dimerization in the cytoplasm;a major determinant for copackaged RNA partner selection is the dimerization initiation signal (DIS);and although RNA transported to the cytoplasm using either the CRM-1 and NXF1 pathway can be packaged efficiently, copackaging of these two RNAs is not favored. We will continue to investigate the mechanisms of viral RNA packaging and RNA trafficking, including the regulation of packaged RNA copy number and partner selection, and HIV-1 RNA nuclear export and trafficking. Additionally, we will modify and redirect RNA packaging specificity and RNA trafficking to gain a better understanding of these processes and how they affect retroviral replication. These studies will shed light on one of the most fundamental features of retroviral replication and will illustrate both the advantages and the limitations of pseudodiploidy. [Corresponds to Hu Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]